Different response of females and males Neotropical catfish (Rhamdia quelen) upon short-term temperature increase.

Climate change has been one of the most discussed topics in the world. Global warming is characterized by an increase in global temperature, also in aquatic environments. The increased temperature can affect aquatic organisms with lethal and sublethal effects. Thus, it is necessary to understand how different species respond to temperature. This study aimed to evaluate how the Neotropical catfish species Rhamdia quelen responds to temperature increases. The fish were exposed to temperatures of 25 °C (control) and 30 °C after gradual temperature increase for 7 days. After 96 h in each temperature, the fish were anesthetized, blood was collected, and after euthanasia, brain, liver, posterior kidney, gills, muscle, and gonads were collected. The gonads were used for sexing, while other tissues were used for the hematological, biochemical, genotoxic, and histopathological biomarkers analysis. Hepatic proteomic analysis with a focus on energy production was also carried out. Blood parameter changes in both sexes, including an increase in glucose in males, leukopenia in females, and genotoxicity in both sexes. Hepatic proteins related to energy production were altered in both sexes, but mainly in males. Others biomarker alterations, such as histopathological, were not observed in other tissues; however, the antioxidant system was affected differently between sexes. These showed that R. quelen juveniles, at temperatures higher than its optimum temperature such as 30 °C, has several sublethal changes, such as hematological alterations, antioxidant system activation, and energetic metabolism alteration, especially in males. Thus, short-term temperature rise can affect females and males of R. quelen differently.

Distribution of Na+/K+-ATPase-immunoreactive ionocytes varies between two superorders of ray-finned fish: Ostariophysi and Acanthopterygii.

Ray-finned fishes of the superorder Ostariophysi are primarily freshwater (FW), and normally stenohaline. Differently, fishes of the superorder Acanthopterygii are essentially marine, and frequently euryhaline, with some secondary FW. Na+/K+-ATPase-immunoreactive ionocytes were localized in the branchial epithelia of 4 species of Ostariophysi and 3 of Acanthopterygii. The Ostariophysi grass carp (Ctenopharyngodon idella, Cypriniformes), twospot Astyanax (Astyanax bimaculatus) and piracanjuba (Brycon orbignyanus), Characiformes, and the jundiá (Rhamdia quelen, Siluriformes), all from FW, displayed ionocytes in the filament plus secondary lamellae (F + SL). In their turn, all the three species of Acanthopterygii showed immunoreactive ionocytes in the filaments only (F). They were the Nile tilapia (Oreochromis niloticus, Cichliformes) in FW, the dog snapper (Lutjanus jocu, Perciformes) in seawater (SW), and the green puffer (Sphoeroides greeleyi, Tetraodontiformes) in SW. Ionocytes normally extend their distribution to the secondary lamellae (F + SL) in Ostariophysi. In Acanthopterygii, we find more plasticity: ionocytes are more frequently restricted to the filament in SW, but also spread to SL in FW. It may be that the occurrence of ionocytes in SL is the ancestral condition, but some euryhaline acanthopterygians rely on the space of the SL for placement of additional ionocytes when in FW absorbing salt. Our study contributed to the identification of the pattern of ionocyte distribution in gills of Ostariophysi in respect to that of Acanthopterygii.

Osmo-ionic regulation and carbonic anhydrase, Na+/K+-ATPase and V-H+-ATPase activities in gills of the ancient freshwater crustacean Aegla schmitti (Anomura) exposed to high salinities.

Aeglidae anomuran crabs originated in the sea, but invaded and diversified in southern South American freshwater (FW) streams. We here aimed at examining their tolerance of increased salinity, after a long time of evolution in FW (~33 million years). Aegla schmitti were exposed to FW and dilute seawater of salinities 15, 20, and 25‰ for 1, 5 and 10 days. Mortality in 35‰ was also assessed. Hemolymph osmolality, Na+, K+, Cl-, and Mg2+ ions, and hydration levels of the abdominal muscle were assayed. The activities of the Carbonic Anhydrase (CA), Na+/K+-ATPase (NKA) and V-H+-ATPase (VHA) were also assayed in the gills. A. schmitti preserves osmoregulatory mechanisms of its marine ancestors. It is able to survive in high salinities (25‰) for at least 10 days. Mortality in 35‰ was of 56% after 1 day, and of 100% after 7 days. In 25‰, NaCl is apparently hyporegulated at all times, while hemolymph osmolality rises after 5 days. CA and NKA activities remained unchanged in all experimental conditions, while VHA activity decreased after 10 days in 25‰. Hemolymph NaCl data was compatible with either hyporegulation and/or putative influx of NaCl into cells for regulatory volume increase (RVI). Further studies should deepen the understanding of the roles of low permeabilities and saturation of high affinity uptake systems in truly FW decapods, in their responses to high salinities. Moreover, the fate of extracellular NaCl as secretion in true hypo-regulation and/or influx into cells for RVI should also be investigated.

Integrated biomarker response index to assess toxic effects of environmentally relevant concentrations of paracetamol in a neotropical catfish (Rhamdia quelen).

The nonsteroidal anti-inflammatory drugs (NSAIDs) are amongst the most commonly detected classes of pharmaceuticals in freshwater environments, with paracetamol being the most abundant. The aim of this study was to evaluate the possible toxic effects of environmentally relevant concentrations (0.25, 2.5 and 25 µg.L-1) of paracetamol in Rhamdia quelen fish exposed for 14 days using different biomarkers. The total count of leukocytes and thrombocytes was reduced at the highest concentration. In the gills, all concentrations of paracetamol reduced the glutathione S-transferase (GST) activity and the reduced glutathione (GSH) levels compared to the control group. The activity of catalase (CAT) was not altered and glutathione peroxidase (GPx) activity increased at the highest concentrations. The superoxide dismutase (SOD) activity decreased at 25 µg.L-1 and the LPO levels increased at 2.5 µg.L-1 when compared to the control group. The concentration of ROS was not different among the groups. In the posterior kidney the activities of GST (2.5 µg.L-1), CAT (2.5 µg.L-1 and at 25 µg. L-1) and GPx and GSH levels increased at all concentrations when compared to the control group. The SOD activity and LPO levels did not change. Paracetamol caused genotoxicity in the blood and gills at concentrations of 2.5 µg.L-1 and in the posterior kidney at 2.5 and 25 µg.L-1. An osmoregulatory imbalance in plasma ions and a reduction in the carbonic anhydrase activity in the gills at 0.25 µg.L-1 were observed. Histopathological alterations occurred in the gills of fish exposed to 25 µg.L-1 and in the posterior kidney at 0.25 and 25 µg.L-1 of paracetamol. The integrated biomarker index showed that the stress caused by the concentration of 25 µg.L-1 was the highest one. These results demonstrated toxic effects of paracetamol on the gills and posterior kidneys of fish, compromising their physiological functions and evidencing the need for monitoring the residues of pharmaceuticals released into aquatic environment.

A systematic evaluation on the relationship between hypo-osmoregulation and hyper-osmoregulation in decapods of different habitats.

Decapods occupy all aquatic, and terrestrial and semi-terrestrial environments. According to their osmoregulatory capacity, they can be osmoconformers or osmoregulators (hypo or hyperegulators). The goal of this study is to gather data available in the literature for aquatic decapods and verify if the rare hyporegulatory capacity of decapods is associated with hyper-regulatory capacity. The metric used to quantify osmoregulation was the osmotic capacity (OC), the gradient between external and internal (hemolymph) osmolalities. We employ phylogenetic comparative methods using 83 species of decapods to test the correlation between hyper OC and hypo OC, beyond the ancestral state for osmolality habitat, which was used to reconstruct the colonization route. Our analysis showed a phylogenetic signal for habitat osmolality, hyper OC and hypo OC, suggesting that hyper-hyporegulators decapods occupy similar habitats and show similar hyper and hyporegulatory capacities. Our findings reveal that all hyper-hyporegulators decapods (mainly shrimps and crabs) originated in estuarine waters. Hyper OC and hypo OC are correlated in decapods, suggesting correlated evolution. The analysis showed that species which inhabit environments with intense salinity variation such as estuaries, supratidal and mangrove habitats, all undergo selective pressure to acquire efficient hyper-hyporegulatory mechanisms, aided by low permeabilities. Therefore, hyporegulation can be observed in any colonization route that passes through environments with extreme variations in salinity, such as estuaries or brackish water.

First Insights into Body Localization of an Osmoregulation-Related Cotransporter in Estuarine Annelids.

The expression of the Na+-K+-2Cl- cotransporter (NKCC), widely associated with cell volume regulation, has never been directly demonstrated in annelids. Its putative presence was firstly recovered in silico, and then using immunofluorescence, its signal was retrieved for the first time in different tissues of four species of estuarine annelids from southern Brazil that are regularly subjected to salinity fluctuations. We tested two euryhaline species (wide salinity tolerance), the nereidids Alitta yarae and Laeonereis acuta (habitat salinity: ~10-28 psu), and two stenohaline species (restricted salinity tolerance), the nephtyid Nephtys fluviatilis (habitat salinity: ~6-10 psu), and the melinnid Isolda pulchella (habitat salinity: ~28-35 psu). All four species showed specific immunofluorescent labelling for NKCC-like expression. However, the expression of an NKCC-like protein was not homogeneous among them. The free-living/burrowers (both euryhaline nereidids and the stenohaline nephtyid) displayed a widespread signal for an NKCC-like protein along their bodies, in contrast to the stenohaline sedentary melinnid, in which the signal was restricted to the branchiae and the internal tissues of the body. The results are compatible with NKCC involvement in cell volume, especially in annelids that face wide variations in salinity in their habitats.

Temperature rise and its influence on the toxic effects caused by cyanotoxins in a neotropical catfish.

Potentially toxic cyanobacterial blooms (cyanoHABs) have become a problem in public water supply reservoirs. Temperature rise caused by climate change can increase the frequency and intensity of blooms, which may influence the cyanotoxins concentration in the environment. This study aimed to evaluate the effect of the temperature on the responses of a Neotropical catfish exposed to a neurotoxin-rich cyanobacterial crude extract (Raphidiopsis raciborskii T3). Juveniles of Rhamdia quelen were exposed to four treatments, based on study data: control at 25 °C (C25), control at 30 °C (C30), crude extract equivalent to 105 cells.mL-l of R. raciborskii at 25 °C (CE25) and 30 °C (CE30). After 96 h of exposure, the fish were anesthetized and blood was taken. After euthanasia, the gill, posterior kidney, brain, muscle, liver and gonad were sampled for hematological, biochemical, genotoxic and histopathological biomarker analysis. Liver was sampled for proteomic analysis for identification of proteins related to energy production. Water samples were collected at the beginning and the end of the experiment for neurotoxins quantification. Different parameters in both males and females were altered at CE25, evidencing the effects of neurotoxins in freshwater fish. At CE30, a water warming scenario, more effects were observed in females than at 25 °C, such as activation of saxitoxin metabolism pathway and genotoxicity. More damage to macromolecules was observed in females at the higher temperature, demonstrating that the increase in temperature can aggravate the toxicity of neurotoxins produced by R. raciborskii T3.

Monitoring water quality in reservoirs for human supply through multi-biomarker evaluation in tropical fish.

Paraíba do Sul River is located at a very densely inhabited region of Brazil crossing the three most industrialized states of the country (São Paulo, Minas Gerais and Rio de Janeiro states). As a result, industrial and farming residues as well as urban sewage are frequently disposed without appropriate treatment. The current study aimed at investigating the water quality in three reservoirs along the Paraíba do Sul River (Ilha dos Pombos, Santa Cecília and Santa Branca), through physiological, morphological, biochemical, and genetic biomarkers. The bioindicator chosen was the catfish Pimelodus maculatus, sampled during the dry (June 2008) and rainy (February 2009) seasons. Also, some water physicochemical parameters were analyzed from the sampling sites, but displayed no alterations according to the Brazilian Agency for Water Quality Legislation. Branchial carbonic anhydrase activity was inhibited in the dry season, while renal carbonic anhydrase activity was inhibited in the rainy season in the Santa Branca reservoir, indicating disturbance of osmoregulatory and acid-base regulation processes. Histopathological alterations were observed in the gills (neoplasic and tissue hyperplasia processes) and liver (necrosis), indicating serious damage to the functional integrity of these organs. A high incidence of melanomacrophage centers was observed in the liver, suggesting an intense immune response in all reservoirs. Acetylcholinesterase and catalase activity showed also differences corroborating some morphological results. Likewise, the induction of the micronucleus and DNA damage indicate genotoxicity, but mainly in the Santa Branca reservoir. Thus, the health status of P. maculatus warrants caution in the use of the water from the 3 reservoirs for direct human consumption, particularly after the accidental spill of endosulfan in November 2008, three months before the rainy season sampling.

Finding Aquaporins in Annelids: An Evolutionary Analysis and a Case Study.

Aquaporins (AQPs) are a family of membrane channels facilitating diffusion of water and small solutes into and out of cells. Despite their biological relevance in osmoregulation and ubiquitous distribution throughout metazoans, the presence of AQPs in annelids has been poorly investigated. Here, we searched and annotated Aqp sequences in public genomes and transcriptomes of annelids, inferred their evolutionary relationships through phylogenetic analyses and discussed their putative physiological relevance. We identified a total of 401 Aqp sequences in 27 annelid species, including 367 sequences previously unrecognized as Aqps. Similar to vertebrates, phylogenetic tree reconstructions clustered these annelid Aqps in four clades: AQP1-like, AQP3-like, AQP8-like and AQP11-like. We found no clear indication of the existence of paralogs exclusive to annelids; however, several gene duplications seem to have occurred in the ancestors of some Sedentaria annelid families, mainly in the AQP1-like clade. Three of the six Aqps annotated in Alitta succinea, an estuarine annelid showing high salinity tolerance, were validated by RT-PCR sequencing, and their similarity to human AQPs was investigated at the level of "key" conserved residues and predicted three-dimensional structure. Our results suggest a diversification of the structures and functions of AQPs in Annelida comparable to that observed in other taxa.

Multiple biomarkers response in a Neotropical fish exposed to paralytic shellfish toxins (PSTs).

The Alagados Reservoir (Southern Brazil) is used as water supply, and since 2002 there have been reports with a presence of cyanobacterial blooms and cyanotoxins. In order to assess the water quality and the ecological integrity of the reservoir, we evaluated biochemical, genotoxic and osmoregulatory biomarkers in the freshwater cichlid fish (Geophagus brasiliensis) that were exposed to PSTs. The fish were sampled in the Alagados Reservoir in February 2016 (Summer) and were divided in three groups: 1) Reservoir group (RES): fish were collected immediately after sampling; 2) Depuration group (DEP): fish were submitted to the depuration experiment for 90 days in the laboratory; and 3) Reproduction group (REP): fish were kept in the laboratory until the fertilization and the chemical analyses were performed on the offspring (F1 generation). In the RES and DEP the blood, brain, muscle, liver and gills were collected for biochemical, genotoxic and osmoregulatory biomarkers analysis. Our results showed that the fish from the Alagados Reservoir (RES) presented oxidative stress and DNA damage; and after 90 days (DEP), the antioxidant system and DNA damage were recovered. Although PSTs were considered a risk to the ecological integrity of this water body; PSTs concentrations were not found in the tissues of the F1 generation. In addition, the biomarkers used were useful tools to evaluate the effects of environment contamination. Therefore, it is necessary to develop new technologies and monitoring programs in order to reduce cyanobaterial blooms, cyanotoxins and human activities that cause the contamination in aquatic environments.

Muscle water control in crustaceans and fishes as a function of habitat, osmoregulatory capacity, and degree of euryhalinity.

This study aimed at detecting possible patterns in the relationship between Anisosmotic Extracellular Regulation (AER) and Isosmotic Intracellular Regulation (IIR) in crustaceans and teleost fish from different habitats and evolutionary histories in fresh water (FW), thus different osmoregulatory capabilities, and degrees of euryhalinity. Crustaceans used were the hololimnetic FW Aegla schmitti, and Macrobrachium potiuna, the diadromous FW Macrobrachium acanthurus, the estuarine Palaemon pandaliformis and the marine Hepatus pudibundus; fishes used were the FW Corydoras ehrhardti, Mimagoniates microlepis, and Geophagus brasiliensis, and the marine-estuarine Diapterus auratus. The capacity for IIR was assessed in vitro following wet weight changes of isolated muscle slices incubated in anisosmotic saline (~50% change). M. potiuna was the crustacean with the highest capacity for IIR; the euryhaline perciforms G. brasiliensis and D. auratus displayed total capacity for IIR. It is proposed that a high capacity for IIR is required for invading a new habitat, but that it is later lost after a long time of evolution in a stable habitat, such as in the FW anomuran crab A. schmitti, and the Ostariophysian fishes C. ehrhardti and M. microlepis. More recent FW invaders such as the palaemonid shrimps (M. potiuna and M. acanthurus) and the cichlid G. brasiliensis are euryhaline and still display a high capacity for IIR.

Late rise in hemolymph osmolality in Macrobrachium acanthurus (diadromous freshwater shrimp) exposed to brackish water: Early reduction in branchial Na+/K+ pump activity but stable muscle HSP70 expression.

Some Macrobrachium shrimps (Caridea, Palaemonidae) are diadromous; freshwater adults are truly euryhaline, while larvae need saline water for development. Branchial Na+/K+-ATPase (NKA) and carbonic anhydrase (CA) are involved in NaCl absorption in freshwater. This study aimed at verifying the time course of the osmoregulatory response of adult Macrobrachium acanthurus to high salinity brackish water (20‰), from the first 30min to 5days. The goal was to detect possible transition from hyper- to hyporegulation, the putative involvement of branchial NKA and CA, or the induction of muscular HSP70 expression. Hemolymph osmotic and ionic concentrations remained relatively stable and close to control levels until ~9h of exposure, but later increased consistently (~50%). A fast reduction in NKA activity (3-6h) was observed; these shrimps seem to shut off salt absorption already in the first hours. Later on, especially after 24h, hemolymph concentrations rise but HSP70 expression is not induced, possibly because constitutive levels are already sufficient to prevent protein damage. Time-dependent response mechanisms effective in high salinity brackish water, resulting in salt loading avoidance and suggestive of hyporegulation should be further investigated in decapods that evolutionary invaded freshwater.

Effects of low concentrations of ibuprofen on freshwater fish Rhamdia quelen.

Ibuprofen is a pharmaceutical drug widely used by the global population and it has been found in aquatic ecosystems in several countries. This study evaluated the effects of ibuprofen in environmental concentrations (0, 0.1, 1 and 10 µg/L) on the freshwaterspecies Rhamdia quelen exposed for 14 days. In the posterior kidney, ibuprofen increased glutathione-S-transferase activity in all groups exposed. Furthermore, increased glutathione peroxidase activity and the levels of reduced glutathione in the group exposed to 10 µg/L. Ibuprofen decreased the carbonic anhydrase activity in the posterior kidney in all exposed groups, and increased the activity in the gills in group exposed to 0.1 µg/L. The levels of plasma magnesium increased in groups exposed to 0.1 and 1 µg/L. In the blood, ibuprofen decreased the white blood cell count in groups exposed to 0.1 e 1.0 µg/L. Therefore, these results indicated that ibuprofen caused nephrotoxicity and demonstrated immunosuppressive effect in Rhamdia quelen.

Depuration time and sublethal effects of microcystins in a freshwater fish from water supply reservoir.

Microcystins (MCs) are hepatotoxins that have been considered to be a worldwide problem due the effects that they can cause to environmental and human health systems. The Iraí Reservoir, located in the South of Brazil, is used as a water supply and MCs concentrations have been reported in this ecosystem. This study aimed to determine the MCs concentrations in the Iraí Reservoir and to evaluate the MCs depuration time and the health of Geophagus brasiliensis using biomarkers. Water and fish samples were collected in the Iraí Reservoir from August 2015 to May 2016. Phytoplankton and chemical analyses were conducted using water samples and the fish were divided into two groups; the Immediate Group (IMM) and the Depuration Group (DEP). In the IMM group, the blood, liver, muscle, brain and gills were collected, in order to evaluate the genotoxic, biochemical and chemical biomarkers. The DEP group was used in the depuration experiment for 90 days, and after this period the fish were submitted to the same procedure as the IMM group. Our results suggested that fish accumulated MCs and it may have caused oxidative stress, neurotoxicity and molecular damage. Furthermore, MCs concentrations increased during the depuration time and it resulted in molecular damage over the first 30 days. After 90 days, the recovery of the antioxidant system occurred. The depuration started on the 15th day, however, the toxins were still present in the samples. Therefore, the effects and the persistence of MCs are a risk to environmental systems and human health.

Immunocytochemical localization of V-H(+) -ATPase, Na(+) /K(+) -ATPase, and carbonic anhydrase in gill lamellae of adult freshwater euryhaline shrimp Macrobrachium acanthurus (Decapoda, Palaemonidae).

Physiological (organismal), biochemical, and molecular biological contributions to the knowledge of the osmoregulatory plasticity of palaemonid freshwater shrimps has provided a fairly complete model of transporter localization in their branchial epithelium. Direct immunological demonstration of the main enzymes in the gill epithelia of adult palaemonids is, however, still incipient. The diadromous freshwater shrimp Macrobrachium acanthurus was exposed to increased salinity (25‰ for 24 hr), and its responses at the systemic level were evaluated through the assays of hemolymph osmolality and muscle hydration, and at cellular and subcellular levels through the activity and localization of the V-H(+) -ATPase, the Na(+) /K(+) -ATPase, and the carbonic anhydrase. Results showed an increase in hemolymph osmolality (629 ± 5.3 mOsm/kg H2 O) and a decrease in muscle hydration (73.8 ± 0.5%), comparing values after 24 hr in 25‰ with control shrimps in freshwater (respectively 409.5 ± 15.8 mOsm/kg H2 O and 77.5 ± 0.4%). V-H(+) -ATPase was localized in pillar cells, whereas Na(+) /K(+) -ATPase in the septal cells. The main novelty of this study was that carbonic anhydrase was localized in the whole branchial tissue, in pillar and septal cells. Exposure to high salinity for 24 hr led to no detectable changes in their localization or in vitro activity. Immunolocalization data corroborated the literature and current models of palaemonid gill ion transport. The absence of changes reinforces the need for the constant expression of these enzymes to account for the euryhalinity of these shrimps.

A multibiomarker evaluation of urban, industrial, and agricultural exposure of small characins in a large freshwater basin in southern Brazil.

Iguaçu River is the second most polluted river of Brazil. It receives agrochemicals and contaminants of urban and industrial sources along its course. A multibiomarker approach was employed here to evaluate the health of a small characin (Astyanax spp.) at two sites along the river, sampled during a dry (autumn) and a rainy (spring) season. Biomarkers were condition factor and somatic indices (gonads and liver); genetic damage (comet assay and micronucleus test); enzyme activities such as hepatic catalase (CAT) and glutathione S-transferase (GST), lipoperoxidation (LPO), branchial and renal carbonic anhydrase (CA), acetylcholinesterase (AChE) in the muscle and the brain, histopathology of the liver and gills, and concentrations of polycyclic aromatic hydrocarbons (PAHs) in bile. There were no consistent differences in biomarker responses between the two study sites. Some biomarkers revealed greater potential impact in the rainy season, when increased amounts of contaminants are washed into the river (combined CAT inhibition and LPO increase, CA upregulation). Other biomarkers, however, revealed potential greater impact in the dry season, when contaminants potentially concentrate (GST induction, AChE inhibition, and liver histopathological alterations). Although of a complex nature, field experiments such as this provide rich data for monitoring protocols and assessment of general risk of exposure to pollutants of river systems.

Regulation of muscle hydration upon hypo- or hyper-osmotic shocks: differences related to invasion of the freshwater habitat by decapod crustaceans.

Decapod crustaceans have independently invaded freshwater habitats from the sea/estuaries. Tissue hydration mechanisms are necessary for the initial stages of habitat transitions but can be expected to diminish, as the capacity for extracellular homeostasis increases in hololimnetic species. Six decapod species have been compared concerning the maintenance of muscle hydration in vitro: Hepatus pudibundus (marine); Palaemon pandaliformis (estuarine resident), Macrobrachium acanthurus (freshwater diadromous), and the three hololimnetic Macrobrachium potiuna, Dilocarcinus pagei, and Aegla parana. The effects of inhibitors of potassium channels (barium chloride) and NKCC (furosemide) were evaluated under isosmotic, and respectively hypo- (50% below iso) or hyper- (50% above iso) conditions. There was high muscle hydration control in H. pudibundus with a possible role of NKCC in isosmotic conditions. Shrimps consistently showed small deviations in muscle hydration under anisosmotic conditions; P. pandaliformis has shown evidence of the presence of NKCC; M. potiuna was the species less affected by both inhibitors, under iso- or anisosmotic conditions. In the two hololimnetic crab species, both independent long-time inhabitants of freshwater, while the capacity to deal with hyper-osmotic shock is decreased, the capacity to deal with hyposmotic shock is retained, possibly because of hemolymph dilution during molting in fresh water. D. pagei apparently depends on potassium channels for volume recovery after swelling, whereas A. parana shows some dependence on NKCC to minimize volume loss in hyper-osmotic conditions. Although no molecular screening techniques have been tried here, data point to distinct cell/tissue transport mechanisms acting upon hydration/volume challenges in decapods of different habitats and lineages.

Unidirectional Na(+) and Ca (2+) fluxes in two euryhaline teleost fishes, Fundulus heteroclitus and Oncorhynchus mykiss, acutely submitted to a progressive salinity increase.

Na(+) and Ca(2+) regulation were compared in two euryhaline species, killifish (normally estuarine-resident) and rainbow trout (normally freshwater-resident) during an incremental salinity increase. Whole-body unidirectional fluxes of Na(+) and Ca(2+), whole body Na(+) and Ca(2+), and plasma concentrations (trout only), were measured over 1-h periods throughout a total 6-h protocol of increasing salinity meant to simulate a natural tidal flow. Killifish exhibited significant increases in both Na(+) influx and efflux rates, with efflux slightly lagging behind efflux up to 60% SW, but net Na(+) balance was restored by the time killifish reached 100% SW. Whole body Na(+) did not change, in agreement with the capacity of this species to tolerate daily salinity fluctuations in its natural habitat. In contrast, rainbow trout experienced a dramatic increase in Na(+) influx (50-fold relative to FW values), but not Na(+) efflux between 40 and 60% SW, resulting in a large net loading of Na(+) at higher salinities (60-100% SW), and increases in plasma Na(+) and whole body Na(+) at 100% SW. Killifish were in negative Ca(2+) balance at all salinities, whereas trout were in positive Ca(2+) balance throughout. Ca(2+) influx rate increased two- to threefold in killifish at 80 and 100% SW, but there were no concomitant changes in Ca(2+) efflux. Ca(2+) flux rates were affected to a larger degree in trout, with twofold increases in Ca(2+) influx at 40% SW and sevenfold increases at 100% SW. Again, there was no change in Ca(2+) efflux with salinity, so plasma Ca(2+) concentration increased in 100% SW. As the killifish is regularly submitted to increased salinity in its natural environment, it is able to rapidly activate changes in unidirectional fluxes in order to ensure ionic homeostasis, in contrast to the trout.

The Na+, K+, 2Cl- cotransporter of estuarine pufferfishes (Sphoeroides testudineus and S. greeleyi) in hypo- and hyper-regulation of plasma osmolality.

The pufferfishes Sphoeroides testudineus and Sphoeroides greeleyi are estuarine species that osmoregulate efficiently, but S. testudineus tolerates seawater dilution to a much higher degree than S. greeleyi. This study aimed at testing whether NKCC is involved with their differential tolerance of seawater dilution, through the analysis of in vivo furosemide (NKCC inhibitor) injection both on hypo-regulation (in 35 per thousand salinity) and hyper-regulation (in 5 per thousand salinity). After exposure for 6 h or 5 days to both salinities, blood samples were obtained for determination of plasma osmolality, chloride, sodium and hematocrit, and muscle samples for determination of water content. Furosemide injection led to increased plasma osmolality and sodium in 35 per thousand and decreased osmolality and chloride in 5 per thousand, when compared to saline-injected controls. Furosemide injection led to hematocrit reduction in both salinities, and muscle water content increase in 5 per thousand and decrease in 35 per thousand in S. testudineus. The results are compatible with NKCC working in branchial NaCl secretion in 35 per thousand, in both species, and a higher role in cell volume regulation in blood and muscle cells of S. testudineus, in both salinities, which could partially explain the stronger capacity of S. testudineus to tolerate seawater dilution during low tide.

Metabolic substrates are not mobilized from the osmoregulatory organs (gills and kidney) of the estuarine pufferfishes Sphoeroides greeleyi and S. testudineus upon short-term salinity reduction

The marine-estuarine species of pufferfishes Sphoeroides testudineus and S. greeleyi are very efficient osmoregulators. However, they differ with respect to their tolerance of salinity reduction. During low tide S. testudineus remains in diluted estuarine waters, whereas S. greeleyi returns to seawater (SW). The hypothesis tested here was that the short-term mobilization of metabolic substrates stored in their main osmoregulatory organs would correlate with this differential tolerance. Fishes exposed to 5ë (for 6 h) were compared to those kept in 35ë. Branchial and renal contents of triglycerides, protein and glycogen were evaluated, and total ATPase activity accounted for the tissues' metabolism. Plasma osmolality, chloride and glucose, hematocrit, and muscle water content were also measured. Total triacylglycerol content was higher in S. greeleyi than in S. testudineus in both salinities and in both organs. Kidney glycogen contents were higher in S. greeleyi than in S. testudineus in 5 and 35ë. Total ATPase activity was reduced in 5ë when compared to 35ë in the kidney of S. greeleyi, and was higher in the gills of S. greeleyi than in those of S. testudineus, in both salinities. Upon exposure to dilute SW, both species displayed a similar osmoregulatory pattern: plasma osmolality and chloride were reduced. Again in both species, stability in muscle water content indicated cellular water content control. Although the metabolic substrates stored in the osmoregulatory organs of both species were not mobilized during these short-term sea water dilution events, some differences could be revealed between the two species. S. greeleyi showed more metabolic reserves (essentially triacylglycerols) in these organs, and its gills showed higher total ATPase activity than those S. testudineus.

Os baiacus marinhos Sphoeroides testudineus e S. greeleyi são freqüentadores de estuários e eficientes osmorreguladores. No entanto, eles diferem quanto à sua capacidade de tolerar a redução de salinidade. Durante a descida de maré S. testudineus permanece no estuário com salinidade reduzida e S. greeleyi retorna para a água do mar. A hipótese testada neste estudo foi a de que a mobilização em curto prazo dos substatos metabólicos armazenados nos órgãos osmorregulatórios poderia explicar esta tolerância diferencial a redução de salinidade. Peixes expostos a 5ë (por 6 h) foram comparados com os mantidos em 35ë . O conteúdo branquial e renal de triacilglicerol, proteína e glicogênio foram avaliados, e a atividade ATPásica total foi quantificada para representar o metabolismo do tecido. Osmolalidade, cloreto e glicose plasmáticos, o hematócrito e o conteúdo de água no músculo também foram mensurados. O conteúdo total de triacilglicerol foi maior em S. greeleyi do que em S. testudineus em ambas as salinidades e em ambos os órgãos. O conteúdo renal de glicogênio foi maior em S. greeleyi quando comparado a S. testudineus na duas salinidades. A atividade ATPásica total foi reduzida em 5ë quando comparada a 35ë no rim de S. greeleyi, e foi maior nas brânquias de S. greeleyi quando comparada aos valores para S. testudineus em ambas as salinidades. Após a exposição a água do mar diluída, ambas as espécies apresentaram padrão osmorregulatório similar: osmolalidade e cloreto plasmáticos foram reduzidos. Em ambas as espécies a estabilidade do conteúdo de água no músculo indicou capacidade de regular o conteúdo de água intracelular. Os substratos metabólicos estocados nos órgãos osmorregulatórios de ambas as espécies não foram mobilizados durante a exposição de curto-prazo a diluição da água do mar, porém algumas diferenças foram reveladas entre as duas espécies. S. greeleyi apresentou maior reserva metabólica (essencialmente triacilglicerol) em seus...